Intersegmental Transfer of Sensory Signals in the
Stick Insect Leg Muscle Control System
Wolfgang Stein,1 Ansgar Büschges,2 Ulrich Bässler3
1
Abteilung Neurobiologie, Universität Ulm, D-89069 Ulm, Germany
2
Lehrstuhl für Tierphysiologie, Zoologisches Institut, Universität zu Köln, D-50923 Köln, Germany
3
Chamissostr. 16, D-70193 Stuttgart, Germany
Received 21 December 2005; accepted 20 February 2006
ABSTRACT: Intersegmental coordination during
locomotion in legged animals arises from mechanical
couplings and the exchange of neuronal information
between legs. Here, the information flow from a single
leg sense organ of the stick insect Cuniculina impigra
onto motoneurons and interneurons of other legs was
investigated. The femoral chordotonal organ (fCO) of
the right middle leg, which measures posture and movement of the femur-tibia joint, was stimulated, and the
responses of the tibial motoneuron pools of the other
legs were recorded. In resting animals, fCO signals did
not affect motoneuronal activity in neighboring legs.
When the locomotor system was activated and antagonistic motoneurons were bursting in alternation, fCO
stimuli facilitated transitions from flexor to extensor
activity and vice versa in the contralateral leg. Following pharmacological treatment with picrotoxin, a
blocker of GABA-ergic inhibition, the tibial motoneurons
INTRODUCTION
During locomotion of legged animals, movement patterns are generated by a close interaction between the
control networks of each leg and coordinating mecha-
Correspondence to: W. Stein ([email protected]).
Contract grant sponsor: DFG; contract grant number: Bu857.
Contract grant sponsor: Graduiertenförderung Rheinland-Pfalz.
' 2006 Wiley Periodicals, Inc.
Published online 10 August 2006 in Wiley InterScience (www.
interscience.wiley.com).
DOI 10.1002/neu.20285
of all legs showed specific responses to signals from the
middle leg fCO. For the contralateral middle leg we
show that fCO signals encoding velocity and position of
the tibia were processed by those identified local premotor nonspiking interneurons known to contribute to
posture and movement control during standing and
voluntary leg movements. Interneurons received both
excitatory and inhibitory inputs, so that the response of
some interneurons supported the motoneuronal output,
while others opposed it. Our results demonstrate that
sensory information from the fCO specifically affects
the motoneuronal activity of other legs and that the
layer of premotor nonspiking interneurons is a site of
interaction between local proprioceptive sensory signals
and proprioceptive signals from other legs. ' 2006 Wiley
Periodicals, Inc. J Neurobiol 66: 1253–1269, 2006
Keywords: coordination; posture control; reflex loop;
nonspiking interneurons; Cuniculina impigra
nisms between these networks (Orlovsky et al., 1999;
Pearson, 1995; Cruse et al., 1995). Both mechanical
couplings between the legs via the ground, as well as
intersegmental neuronal pathways, contribute to this
coordination (Dürr et al., 2004). It is unknown which
specific kind of neuronal information is exchanged
between the joint controllers, mainly because investigations of intersegmental influences in active animals
are difficult. Besides receiving neuronal signals from
the neighboring segments, each joint controller produces its own rhythmic motor output that is strongly
regulated by intrasegmental mechanisms. In crustaceans, for example, the thoracic-coxal muscle recep1253
1254
Stein et al.
tor organ influences the motoneurons of other ipsilateral legs in isolated, nonrhythmic preparations (Sillar
et al., 1987). However, these effects elicit suprathreshold activation of the motoneurons only when a
central pattern generator is active. Thus, it is often
difficult to separate the effects of central pattern generators and of local sensory feedback from those
signals arising from the movements of other legs.
Similarly, in stick insects, the femoral chordotonal
organ (fCO) of the front leg affects motoneurons in
the ipsilateral middle leg when the locomotor system
is active (Ludwar et al., 2005). By contrast, in resting
animals, proprioceptive signals from the legs do not
or only weakly influence the motoneurons in the
neighboring segments (Graham and Wendler, 1981;
Cruse et al., 1993; Ludwar et al., 2005). Previous
investigations have therefore focused more on synaptic inputs that intersegmental interneurons receive
from proprioceptive sense organs (Laurent and
Burrows, 1989; Büschges, 1989; Brunn and Dean,
1994) than on the characterization of their output
sites. Here, we examined the influence of a single
sense organ, the fCO, on inter- and motoneurons of
the femur-tibia joints of adjacent legs. While in each
hemiganglion local sensory information from the
fCO is processed by identified local nonspiking interneurons (Büschges, 1990; Sauer et al., 1996; Stein
and Sauer, 1998), which also contribute to the generation of motor activity during adaptive leg reflexes
(Kittmann et al., 1996) and walking (Büschges et al.,
1994), it is unclear whether this premotor network is
also involved in the processing of sensory information from other legs. For our investigation, we
restrained the animals and either aroused them by
touching the abdomen with a paintbrush or we bath
applied picrotoxin (PTX) to decrease GABA-ergic inhibition in the thoracic nerve cord. PTX does not
affect the membrane properties of tibial inter- and
motoneurons (Sauer et al., 1997), but it drastically
enhances sensory inputs to these (local) neurons. We
show that PTX also enables the intra- and intersegmental transmission of specific fCO signals to interand motoneurons of neighboring legs although the
animals remain in an inactive state and do not perform active leg movements. We discuss the putative
functional relevance of the observed effects regarding
the generation of coordinated leg movements like
walking or rocking.
lum impigrum Brunner) from the colonies at Kaiserslautern
and Ulm. Experiments were carried out in accordance with
the European Communities Council Directive of 24 November 1986 (86/609/EEC) and with the guidelines laid
down by the NIH in the US regarding the care and use of
animals for experimental procedures.
Preparation and Recordings
Extracellular recordings of motoneurons, electromyograms
(EMGs), and intracellular recordings from extensor motoneurons and nonspiking interneurons of the mesothoracic
ganglion were performed as described in detail in previous
studies (Büschges, 1990; Sauer et al., 1995). In brief, the
animals were opened dorsally, the gut was removed, and
the body cavity filled with stick insect saline (Weidler and
Diecke, 1969; Bässler, 1977). The mesothoracic ganglion
was fixed to a wax-coated ganglion holder and the ganglion
sheath was treated with Pronase E (Merck KGaA, Darmstadt, Germany). The activities of inter- and motoneurons
were recorded in the dorso-lateral neuropil region of the
mesothoracic ganglion. Glass microelectrodes were filled
with 2 M KAc (tip resistance: 10–20 MO) or 1 M LiCl
(shaft solution) and 5% Lucifer Yellow (tip solution; tip resistance: 40–70 MO). Recorded neurons were identified
both by morphological and physiological properties
(Büschges, 1990, 1994; Driesang and Büschges, 1993;
Sauer et al., 1996; Stein and Sauer, 1998). Extracellular
recordings of the extensor nerve F2 (containing the slow extensor tibiae motoneuron SETi, the fast extensor tibiae
motoneuron FETi, and the common inhibitor 1 motoneuron
CI1), the protractor nerve nl2, and the retractor nerve nl5
were obtained using paraffin-oil hook electrodes (Schmitz
et al., 1991).
Force measurements were obtained by attaching the
force transducers (Swema SG.02109 with Hellige TF119
bridge) to the proximal part of the tibiae of the different
legs, thus giving a relative measurement of the combined
forces exerted by extensor and flexor tibiae muscles.
Experiments were performed during daylight. Under
these conditions resting animals adopted an inactive behavioral state in which no spontaneous bursts of motoneuron
activity could be detected and resistance reflexes were elicited in the local joint control system (Bässler, 1993). For
measurements in the active animal, we touched the abdomen of the animal with a paintbrush until extensor and
flexor motoneurons produced bursts of action potentials
(Bässler and Büschges, 1998), indicating that the animal
had adopted the active behavioral state (Driesang and
Büschges, 1996).
Mechanical Stimulation of the fCO
MATERIALS AND METHODS
All experiments were carried out on adult female stick
insects Cuniculina impigra Redtenbacher (synonym BacuJournal of Neurobiology. DOI 10.1002/neu
The fCOs of the right and left middle legs were mechanically stimulated by inserting the receptor apodeme into a
stimulus clamp, and cutting it distally to the clamp. The
stimulus clamp was moved by a pen-motor (Hellige), which
was controlled by a function generator able to produce
Interleg Couplings in the Stick Insect
ramp-and-hold stimuli of different velocities (corresponding to tibial movements with velocities between 7 and
12408/s; Weiland and Koch, 1987) and different holding
times. In some experiments, sinusoidal stimuli of different
frequencies were also used. The standard stimulation amplitude was 400 m (simulating a movement of the tibia of
approximately 408; Weiland et al., 1986). The starting position of the fCO apodeme was fixed at a femur-tibia angle of
1008, unless mentioned otherwise. In all experiments, the
FT-feedback loops of all legs were opened by either cutting
the fCO receptor apodemes or ablating the leg (see below).
The fCO of the right middle leg was stimulated and the
responses of the extensor motoneurons of the left foreleg
(L1), the right foreleg (R1), the left middle leg (L2), the
right middle leg (R2), the left hind leg (L3), and the right
hind leg (R3) were recorded extracellularly. In some experiments force transducers were attached to the stump of the
tibia instead of extracellular electrodes. In experiments in
which the responses of only the two middle legs were
investigated, all other legs were ablated either in the middle
of the coxa or in the proximal femur to prevent an influence
of the sense organs of these legs. In these experiments, the
activity of the flexor muscle of L2 was measured with EMG
recordings.
1255
same animal were compared. The Mann-Whitney U test
was used to test nonparametric statistical significance. Values are given as mean 6 SD or as median plus interquartile
range (iqr). Regression lines were calculated according to
the least squares regression equation. Square correlation
coefficient (r2) and its difference from zero are given. The
F-distribution was used to test significant differences of r2
from zero. In the following, N gives the number of animals,
while n gives the number of trials. For all statistical tests,
significance was indicated in figures using the following
symbols: *p < 0.05; **p < 0.01; ***p < 0.001. Peri-stimulus time histograms always represent averaged data.
RESULTS
We investigated the information transfer of sensory
signals between the joint control systems of different
legs by focusing on a single leg joint, the femur-tibia
(FT) joint. A single proprioceptor, the fCO, measures
the position and the movement of the tibia. We stimulated the fCO of one leg and measured the responses
of the motoneuron pools of the FT-joints of other
legs.
Pharmacological Treatment
PTX (Sigma) was diluted to a concentration of 102 M in
dimethyl sulfoxide (Sigma) and stored as a stock solution.
Prior to each experiment, the stock solution was further
diluted in saline to a concentration of 105 M (Sauer et al.,
1997). One to two milliliters was then added to the bath.
Because the bath volume (1–2 mL) varied between animals,
the final concentration could only be estimated to be about
5 106 M. Concentration values thus do not represent the
final concentration, but rather the concentration of PTX that
was applied to the bath. Application of dimethyl sulfoxide
itself at the appropriate concentration had no effects on the
neuronal activity. The effects of PTX were irreversible,
even after more than 1 h of saline perfusion. This has also
been reported in another study on stick insects (Sauer et al.,
1997) and is probably due to the slow access of pharmacological substances, like PTX and other solutions, to the neuropil region in the thoracic ganglia (A.E. Sauer and
H. Kohl, personal communication) as a result of the effective isolation of the nervous system by the neurolemma
(Dörr et al., 1996; Treherne, 1985). In general, the reversibility of PTX effects decreases with higher concentration
and longer exposure, also common in other systems (M.P.
Nusbaum, personal communication). In each of our experiments the nervous system was exposed to PTX for up to 1 h
and 30 min.
Statistics
Statistical significance between means was calculated
according to a modified t test (Dixon and Massey, 1969) or
with a paired samples t test when observations from the
Stimulation of the Contralateral fCO
Facilitates Transitions from Extensor to
Flexor Activity and Vice Versa in Active
Animals
The fCO of the right middle leg (R2) of the restrained
animal was stimulated with ramp-and-hold stimuli
while the responses of the tibial motoneurons in the
contralateral leg were recorded. No stimulus related
responses were observed in inactive animals
[example shown in Fig. 1(G), 3]. In contrast, in active
animals (see Materials and Methods), fCO stimulation facilitated transitions in tibial motoneuron activity from flexor to extensor activity and vice versa in
the contralateral leg. Transitions in both directions
were elicited by either stimulus direction. Figure
1(A–D) shows example recordings for transitions
from flexor to extensor activity [Fig. 1(A)] and extensor to flexor activity [Fig. 1(B)] during elongation of
the fCO, and transitions from flexor to extensor activity [Fig. 1(C)] and extensor to flexor activity [Fig.
1(D)] during relaxation of the fCO. The median burst
duration of extensor and flexor motoneurons was significantly shorter when fCO-stimuli were applied in
comparison to bursts without fCO stimulation [Fig.
1(E) left; median burst duration with stimulation 1.83
s, iqr 1.00–3.03 s, n ¼ 1368; median burst duration
without stimulation 2.00 s, iqr 1.05–4.09 s, n ¼ 331,
p < 0.01].
Journal of Neurobiology. DOI 10.1002/neu
1256
Stein et al.
The effect of fCO stimulation was a result of the
phasic ramp stimulus rather than a tonic increase of
excitation caused by the stimulus because the durations of the bursts depended on when the stimuli were
started during the bursts. When stimuli were applied
during the first half of the burst, the median burst duration was 1.96 s (iqr 1.04–3.41 s, n ¼ 662) and thus
not different from the burst duration of bursts without
fCO stimuli. However, when stimuli were applied
during the second half of the burst, the median burst
duration was significantly shorter (median 1.73 s, iqr
0.95–2.89 s, n ¼ 706, p < 0.01), indicating that stim-
uli were effective when presented late in the burst.
Indeed, we found a correlation between mean burst
duration and stimulus phase (r2 ¼ 0.34, slope is nonzero, p < 0.01, n ¼ 1368). With increasing stimulus
phase, burst durations decreased, indicating that stimuli were more likely to end a burst the later they were
presented in the burst (stimulus phase was calculated
by normalizing the stimulus time during a burst to the
burst duration; mean stimulus durations were calculated for phase BINs of 0.05).
To provide further support for the hypothesis that
fCO stimulation elicited transitions of motor activity,
we compared the delay between stimulus and the end
of a burst with the half duration of the bursts. The
half duration would be the average predicted value
for the delay between stimulus and the end of the
burst if stimuli fell randomly within bursts and had
no effect on burst duration. In this case, stimuli would
not cause transitions in motor activity. We found,
however, that the delay (median 0.72 s, iqr 0.29–
1.62 s, n ¼ 1368) was significantly shorter than
the half duration [median 0.91 s, iqr 0.50–1.51 s, n ¼
1368, p < 0.01; Fig. 1(E) right], which suggests that
stimuli, indeed, supported activity transitions.
Figure 1 Sensory signals from a single leg sense organ
affect the motoneuron pools of the contralateral leg when
the locomotor system is activated and in intermediate states
of activity. (A) In active animals, fCO elongation increased
the probability for transitions from flexor to extensor activity. CI1, common inhibitor 1 motoneuron. Flexor activity
was recorded as EMG. (B) fCO elongation terminated extensor activity and facilitated the beginning of a flexor
burst. (C) fCO relaxation ended a flexor burst and elicited
extensor activity. (D) fCO relaxation caused a transition
from an extensor burst to flexor activity. (E) Left: plot of
burst duration without fCO stimulation (n) and with fCO
stimulation (s). Interquartiles and median are given. n ¼
331 bursts without fCO stimulation, n ¼ 1368 with fCO
stimulation. Right: comparison of delay between start of
fCO stimulus and end of burst (d; n ¼ 1368) and half duration of bursts (h; n ¼ 1368). (F) Distribution of delays
between stimulus and end of burst with (black; n ¼ 1368)
and without (grey; n ¼ 331) stimulus connected to the fCO.
For the latter experiments, stimuli were applied randomly
during the bursts. Data were normalized to the number of
bursts. Bin width: 50 ms. (G) Flexor (EMG) and extensor
motoneurons (extracellular nerve recording) in L2 slowly
changed their response from stimulus-induced transitions
between the two motoneuron pools (1) to stimulus-induced
excitation causing coactivation of extensor and flexor motoneurons (2). Eventually, they stopped responding (3). Asterisk indicates largest units were clipped. Note that amplitude
scaling in (2) and (3) is different from (1) to better show the
neuronal response.
Journal of Neurobiology. DOI 10.1002/neu
Interleg Couplings in the Stick Insect
1257
then activated by elongation and relaxation of the
contralateral fCO, or by either of the two stimuli (this
differed within one animal and between different animals). This influence of fCO signals on the contralateral motoneurons considerably weakened and disappeared [Fig. 1(G), 3] during the following 177 6 112 s
(N ¼ 7).
Figure 2 Bath application of picrotoxin (PTX, 105 M)
establishes stimulus-specific interleg couplings. Simultaneous recordings of relative (total) tibial forces (L1, L2, L3,
R3), and extracellular recordings of the extensor nerves in
R1 and R2 during sinusoidal stimulation of the fCO. Left:
in saline, no stimulus-correlated nerve activities or forces
were observed, except for SETi response in the local leg
(R2, arrow: resistance reflex). After PTX/application the
FT-loops of all legs responded to the stimulus. L1–3, left
legs; R1–3, right legs; 1 ¼ foreleg, 2 ¼ middle leg, 3 ¼
hind leg. Asterisk indicates crosstalk from flexor muscle.
Because stimuli decreased the burst duration, one
would expect the burst end to occur within a shorter
time period after the stimulus than for stimuli that did
not affect the burst duration. We tested this hypothesis by comparing the distribution of delays with that
obtained from blind experiments in which stimuli
were applied randomly and were not connected to the
fCO [Fig. 1(F)]. With the stimulus connected to the
fCO, transitions in motor activity were more likely to
occur within a delay of 200 ms after the stimulus in
comparison to the blind control. For these transitions,
the median burst duration (0.83 s, iqr 0.55–1.54 s,
n ¼ 238) was significantly shorter than the burst duration of all bursts (median 1.83 s, iqr 1.00–3.03 s, n ¼
1368, p < 0.01).
When we stopped arousing the animal with the
paintbrush, a gradual change from the active to the
inactive state was observed [N ¼ 7; example shown
in Fig. 1(G)] in the same way as has been described
previously (review in Bässler, 1983, 1993). Immediately after the end of the paintbrush stimulation, extensor and flexor motoneurons still produced bursts
of action potentials. fCO stimulation of the contralateral leg elicited transitions between extensor and
flexor discharge [Fig. 1(G), 1]. During ongoing fCO
stimulation this response disappeared through a phase
of coactivation in tibial motoneurons along with a
cessation of bursting activity in tibial motoneurons
[Fig. 1(G), 2]. Extensor and flexor motoneurons were
Application of PTX Unmasks the Transfer
of Sensory Information from the fCO to
the Tibial Motoneurons of Other Legs in
Inactive, Restrained Preparations
The preceding experiments have shown that in the
active animal information from the fCO can affect
the activity of motoneurons supplying the contralateral leg. In active animals, however, it is difficult to
investigate whether and how information from sensory organs in different legs is processed in the local
joint control systems. We thus chose to examine this
in pharmacologically treated animals. We bath
applied PTX (105 M; see Sauer et al., 1997) and
subsequently assayed the influence of signals from
the fCO of the right middle leg (R2) on the activity of
tibial motoneurons and muscles in the adjacent legs
in restrained and inactive animals. The fCO was
stimulated sinusoidally. In saline, only the motoneurons of the stimulated leg responded to the stimulus. A
resistance reflex was generated (Fig. 2, left, arrow,
summary: Bässler, 1993). In PTX, not only the motoneurons of the stimulated leg, but also the muscles
and motoneurons of all other FT joints showed stimulus-related responses (Fig. 2, right). The activities of
the extensor motoneurons in the right foreleg (R1)
and in R2, and the forces moving the tibia of the left
foreleg (L1), the left middle leg (L2), the left hind leg
(L3), and the right hind leg (R3) were recorded simultaneously. All FT loops were in an open-loop condition with the receptor apodemes of all fCOs cut.
Thus, no sensory feedback from the local FT joints
affected the recordings.
In N ¼ 12 of 15 tested animals, either stimuluscorrelated motoneuronal activity or tibial forces
appeared in the neighboring segments 17.6 6 6.9 min
after PTX application (N ¼ 12). In three animals we
observed no response, either in the muscles or in the
motoneurons of the unstimulated legs.
In 10 of the 12 animals, the activities in the FT
loops were such that the legs of one segment would
have moved in different directions: when a flexion
force was exerted at the tibia of L1, the activity of the
extensor motoneurons in R1 increased. In the mesothoracic segment, the stimulated leg R2 produced reJournal of Neurobiology. DOI 10.1002/neu
1258
Stein et al.
sistance reflexes, which means that, for example, a
signaled flexion of the right leg (elongation of the
fCO) would elicit an extension of this leg. The same
stimulus caused a flexion force in the contralateral
left leg. Relaxation of the fCO elicited opposite
results. The tibial muscles of the hind legs also produced forces that antagonized each other; when an
extension force at the tibia of R3 occurred, a flexion
force at the tibia of L3 was recorded. In two of the 12
responding animals, only the contralateral leg control
system was influenced by fCO stimulation in the
presence of PTX.
Characterization of the Sensory
Information, which Is Transmitted to the
Tibial Motoneurons of Other Legs in the
Presence of PTX
To characterize the influence of fCO stimulation in
R2 on the tibial motoneurons of the neighboring legs
in PTX, we applied ramp-and-hold stimuli to the
fCO. Ramp-and-hold stimuli consist of a velocity
component (the ramp) and a position component (the
hold phase) and thus allow separation of these two
movement parameters. Figure 3(A) shows a recording
of the contralateral extensor motoneurons along with
an EMG recording of the flexor muscle in this leg
during stimulation of the ipsilateral fCO. In saline no
response was observed in resting animals. After PTX
application SETi was tonically active (see also Sauer
et al., 1997). The activity of both extensor motoneurons and the activity of the flexor muscle increased
during elongation as well as relaxation of the fCO.
This response gradually increased with time after
PTX application. During the hold phase, the activity
of the extensor motoneurons was lower than before
stimulus onset. Because the flexor muscle was activated along with the extensor motoneurons during the
ramp part of the fCO stimulus, a leg movement would
be the result of a cocontraction of extensor and flexor
muscles. For sinusoidal stimuli, as shown in Figure 2,
the flexion force exerted at the tibia during elongation
of the fCO thus appears to be larger than the extension force, because this stimulus led to a flexion of
the contralateral leg.
The influences of position and velocity signals
from the fCO were investigated in more detail for the
extensor motoneurons of all leg-bearing segments.
Stimulus Velocity. In PTX, the excitatory extensor
motoneurons SETi and FETi of all other legs
responded similarly to ramp-and-hold stimulation of
the fCO in R2. During elongation and/or relaxation
Journal of Neurobiology. DOI 10.1002/neu
Figure 3 (A) PTX slowly established the response of contralateral extensor and flexor tibiae motoneurons to ipsilateral
fCO stimulation. EMG of the flexor tibiae muscle and extracellular recording of SETi and FETi, in saline, 10 and 20 min after
PTX application. CI1, common inhibitor 1 motoneuron. (B)
PSTHs (averaged, BIN width 0.1 s) of the extensor activity
(SETi and FETi) in L2 in saline, and after PTX application:
response type I, II, and III. Scale bars: horizontal, 1 s; vertical,
0.5 spikes/BIN. (C) Comparison of averaged SETi-activity in
all legs during ramp-and-hold elongation of the fCO in R2 with
different stimulus velocities. In all legs stimulus-induced activity changed significantly with stimulus velocity (***p < 0.001,
7 < N < 11, different from values at 508/s). (D) Response of
the flexor motoneurons (EMG) in L2 during ramp-and-hold
stimulation of the contralateral fCO with different stimulus
velocities (25, 45, 2008/s), in PTX. The activity of the flexor
muscle increased with increasing stimulus velocity. Top trace:
PSTH (averaged) of flexor activity, BIN width 0.2 s; middle
trace: EMG sample of flexor muscle; bottom trace: stimulus.
Interleg Couplings in the Stick Insect
1259
Table 1 Responses and Numbers of Recordings of Extensor Motoneurons in Legs R1, L1, L2, L3,
and R3 during Ramp-and-Hold Stimulation of the fCO in R2 during PTX Application
Leg
Response I
Response II
Response III
No Response
Number of Recordings
L1
R1
L2
L3
R3
5
6
23
5
6
4
5
19
6
9
0
0
9
0
0
0
0
8
0
0
9
11
59
11
15
L1–3, left legs; R1–3, right legs; 1 ¼ foreleg, 2 ¼ middle leg, 3 ¼ hind leg.
stimuli either one or both extensor motoneurons were
activated. Three different types of responses were
obtained: (I) both elongation and relaxation of the
fCO caused an activation of the extensor motoneurons [Fig. 3(B), I]; or (II) the extensor motoneurons
were activated only during elongation [Fig. 3(B), II];
or (III) only during relaxation of the fCO [Fig. 3(B),
III]. In the contralateral middle leg L2, 23 out of 59
animals showed response type I, 19 showed response
type II, and nine showed response type III. In the
remaining eight animals, no response was observed.
Similar results were obtained for the other legs, however, only response types I and II occurred (Table 1).
The activities of the extensor motoneurons during the
stimulus ramp depended on stimulus velocity. To test
the response of the extensor motoneurons of all legs,
we used two velocities (50, 2508/s). Generally, with
increasing ramp velocity the activity of the extensor
motoneurons of all legs increased [Fig. 3(C); N ¼ 11
animals, n ¼ 7 stimuli each, p < 0.001 for L1, R1,
L2, and R2; N ¼ 7 animals, n > 6 stimuli each, p <
0.001 for L3 and R3]. Similarly, the activity of the
flexor muscle, as measured by the number of spikes
in EMG recordings of L2, increased significantly with
stimulus velocity [N ¼ 3 animals, n > 7 stimuli, p <
0.001, example shown in Fig. 3(D)].
Stimulus Position. Three different positions of the
fCO stimulus in R2 were tested in increasing and
decreasing sequence to examine the influence of tibial
position on the activities of the extensor motoneurons
of the other legs. We used ‘‘staircaselike’’ stimuli
with holding positions corresponding to 180, 110,
and 208 and measured motoneuron activities during
the hold phases of the stimuli (after the end of the velocity-sensitive response). The activities of the extensor motoneurons of all legs depended on fCO position. The results of 11 experiments in total are summarized in Figure 4. Whereas in R2 strong resistance
reflexes were elicited (compare to Sauer et al., 1997)
and (extensor) motoneuronal activity increased with
more elongated positions of the fCO [Fig. 4(E)], the
contralateral leg showed the opposite response char-
acteristic: with more relaxed fCO positions (signaling
an extended joint), extensor activity increased [Fig.
4(B)]. In L1 and L3 the activity of the extensor motoneurons also increased significantly with more relaxed stimulus positions [extended tibial positions;
Fig. 4(A,C)]. In R1 and R3, on the other hand, more
elongated stimulus positions (flexed joint positions)
Figure 4 In PTX, position information from the local
fCO affects the tibial motoneurons in all other legs. Average of the SETi activity in the different legs elicited by fCO
stimulation in R2, different holding positions (180, 110,
and 208; stepwise stimuli). Averaged SETi activity was dependent on stimulus position in all legs (*p < 0.05; ***p <
0.001; N ¼ 11, 208 differed significantly from 1108; 1108
differed significantly from 1808). (A–C) Left legs (L1–L3).
(D–F) Right legs (R1–R3). 1 ¼ foreleg, 2 ¼ middle leg,
3 ¼ hind leg.
Journal of Neurobiology. DOI 10.1002/neu
1260
Stein et al.
significantly increased extensor motoneuron activity
[Fig. 4(D,F)].
In summary, sensory signals, which indicate flexed
positions of the FT joint in R2, decreased SETi activity in all contralateral legs and increased SETi activity in the ipsilateral legs. This corresponds to the
results shown in Figure 2, in which the contralateral
legs moved approximately in phase with the signaled
movement of the FT joint in R2, while the ipsilateral
legs moved roughly in antiphase.
In the Presence of PTX, Sensory Signals
from the fCO Excite Contralateral
Extensor Tibiae Motoneurons
In a first step to analyze how sensory signals from the
fCO affect the tibial motoneurons of other legs, we
focused on the influence of the fCO on the contralateral extensor motoneurons of the same segment. We
recorded intracellularly from the extensor motoneurons in L2 and stimulated the fCO in R2 with rampand-hold stimuli in saline and after the stabilization
of the PTX effect. In saline in the resting animal,
FETi (N ¼ 8) was not affected by stimulation of the
fCO in R2. FETi was never spontaneously active in
saline. In contrast, in PTX, FETi was spontaneously
active, with a firing frequency of 15.2 6 14 Hz (N ¼ 3).
FETi was depolarized by elongation stimuli delivered
to the fCO in R2 [Fig. 5(A)]. In order to measure relative input resistance, FETi was held hyperpolarized
so that firing was suppressed, and hyperpolarizing
current pulses of 0.5 nA and 100 ms durations were
injected repeatedly. During the stimulus related depolarization, FETi input resistance decreased significantly by 15.2 6 5% (N ¼ 3, p < 0.01) in comparison
to before fCO elongation. The decrease in input resistance during elongation of the contralateral fCO
argues for an increase of excitatory synaptic inputs.
The other excitatory motoneuron innervating the
extensor tibiae, SETi, showed a similar response to
Figure 5 Input from the contralateral fCO specifically
affects tibial motoneurons and premotor interneurons in the
ipsilateral leg. (A) Influence of fCO stimulation in R2 on
the membrane potential of FETi in L2 in saline and PTX
(105 M). In saline, FETi did not respond to stimulation of
the contralateral fCO. In PTX, FETi was spontaneously
active and depolarized during fCO elongation. (B) Influence of fCO stimulation in R2 on the membrane potential
of SETi in L2. Averaged intracellular recording of SETi in
L2 during ramp-and-hold stimulation of the fCO in R2.
SETi was hyperpolarized to prevent spike discharge. Vertical scale bar: 4 mV. (C) Intracellular recording of NSI E7
in L2 during stimulation of the fCO in R2 in saline and in
PTX. Black arrows, depolarization; open arrow, hyperpolarization below resting potential. Vertical scale bar: 1 mV.
(D) Intracellular recording of NSI E5/6 in L2 during stimulation of the fCO in R2 in saline and in PTX. Black arrow,
depolarization during tonic fCO elongation; open arrow,
hyperpolarization below resting potential. Vertical scale
bar: 1 mV. (E) Averaged intracellular recording of NSI I4
in L2 during elongation of the ipsilateral (L2) fCO, in PTX.
(F) Averaged response of NSI I4 in L2 to elongation of the
contralateral (R2) fCO, in PTX. The response to contralateral stimulation always exceeded the response to ipsilateral
stimulation. (G) NSI E8 received inhibition during elongation of the contralateral fCO. Intracellular recording of NSI
E8 in L2 during stimulation of the fCO in R2 after PTX
application. Hyperpolarization (open arrows) increased
with increasing stimulus velocity. Vertical scale bar: 3 mV.
(H) Averaged responses of E8 to elongation of the contralateral fCO, in PTX, revealed that the hyperpolarization
was preceded by a short-latency depolarization (black
arrow). Vertical scale bar: 2 mV.
Journal of Neurobiology. DOI 10.1002/neu
Interleg Couplings in the Stick Insect
fCO signals in R2. In saline at rest, no response to
fCO stimulation was detectable for SETi. SETi was
active with a spontaneous firing frequency of 5.5 6 5
Hz (N ¼ 4). In the presence of PTX, SETi showed an
elevated level of spontaneous activity of 45.2 6 16
Hz (N ¼ 4). SETi was depolarized during elongation
and relaxation of the contralateral fCO [Fig. 5(B)].
The depolarization during fCO elongation was associated with a significant decrease of input resistance
(17.9 6 5.3%, N ¼ 4, p < 0.01) in comparison to the
situation without fCO stimulation. Thus, as in FETi, a
stimulus-related activation of excitatory synaptic
inputs seems very likely.
Local Premotor Nonspiking Interneurons
Process Signals from the fCO of the
Contralateral Leg
SETi and FETi receive synaptic drive from identified
local premotor nonspiking interneurons (NSIs, stick
insect: Büschges, 1990; Sauer et al., 1995, 1996;
Stein and Sauer, 1998). These NSIs are key premotor
elements in the segmental leg motor control systems
of insects (for reviews see Bässler and Büschges,
1998; Burrows, 1996; Field and Matheson, 1998).
We tested whether they are the sites of interaction
between local proprioceptive sensory signals and proprioceptive signals from other legs. We recorded
intracellularly from identified premotor NSIs of the
FT joint to examine whether they are involved in
processing and transmitting sensory signals from the
contralateral fCO onto local motoneurons. Before
application of PTX, NSIs did not respond to stimulation of the contralateral fCO. In PTX, however, all
but one type of the investigated NSIs were affected
by signals from the contralateral fCO. We classified
the responses according to their relative strengths and
signs. The results are summarized in Table 2.
NSIs of type E1 (N ¼ 3), E3 (N ¼ 4), E4 (N ¼ 5),
E7 (N ¼ 4), I1 (N ¼ 6), and I4 (N ¼ 4) were depolarized by both elongation and relaxation of the contralateral fCO. This is exemplified for NSI E7 in Figure
5(C) (black arrows indicate depolarization). NSIs E7
and E1 were additionally hyperpolarized below resting potential during the hold phase of the stimulus
[open arrow in Fig. 5(C)]. Interneuron I2 received
depolarizing signals only during contralateral fCO
elongation, while NSI E2 was not affected at all by
the stimulus. In these two cases, we tonically injected
de- and hyperpolarizing currents under DCC conditions to move the membrane potential up to 20 mV
away from the resting potential. Even then no influence of contralateral fCO elongation (E2) or contra-
1261
lateral fCO relaxation (E2, I2) was found. Interneurons of type E5/6 (N ¼ 5) received depolarizing inputs
during the ramp-and-hold phase of the contralateral
fCO stimulus [Fig. 5(D)]. During relaxation of the
fCO, however, the membrane potential was hyperpolarized below its resting value [open arrow in
Fig. 5(D)]. There was a small position-dependent
depolarization with increasing elongation of the fCO
in four of the five recordings (black arrow). It was
obvious for E4 and I4 that the depolarization amplitudes during contralateral fCO stimulation always
exceeded the depolarization elicited by stimulation of
the ipsilateral fCO, at least for one stimulus direction.
In Figure 5(E,F) the average responses of NSI I4 to
elongation of the ipsilateral fCO [Fig. 5(E)] and the
contralateral fCO [Fig. 5(F)] are shown. The depolarization during elongation of the contralateral fCO
was significantly larger (by 62.3 6 41%, N ¼ 4, p <
0.01) than during stimulation of the ipsilateral fCO.
In contrast to all other NSIs, E8 (N ¼ 5) received
hyperpolarizing inputs during elongation of the contralateral fCO [Fig. 5(G)]. The amplitude of these
inputs increased with stimulus velocity. They thus
antagonized the stimulus-related activation of the
SETi and FETi motoneurons. Because the input
resistance decreased significantly, by 6.79 6 1.1%
(N ¼ 4, p < 0.05), during the ramp phase of fCO elongation, the hyperpolarization appeared to be elicited
by an increase of PTX-insensitive inhibitory synaptic
inputs, rather than by a decrease of tonic excitation. At
high stimulus velocities, the hyperpolarization was
preceded by a short-latency depolarization [Fig. 5(H),
arrow], indicating that excitatory and inhibitory pathways affected E8 in parallel (similar to the processing
of local fCO signals in NSIs; Sauer et al., 1995).
We evaluated the latencies between the start of
fCO elongation and the start of the responses in the
different types of NSIs, for comparison with the
latencies of ipsilateral fCO stimulation. In the stick
insect, NSIs are known to receive short latency (and
thus most likely direct) synaptic inputs from fCO
afferents (Sauer et al., 1996). The latencies for the intracellular response to stimulation of the contralateral
fCO were always significantly longer compared to
the latencies of the responses to ipsilateral stimulation
(Table 2; averaged latency of all NSIs to contralateral
fCO stimulation with a velocity of 2508/s: 21.08 6
2.72 ms, N ¼ 8; averaged latency of all NSIs to ipsilateral fCO stimulation: 9.72 6 3.01 ms, N ¼ 9, significantly different with p < 0.001).
In a different set of experiments we used current
injections into the NSIs to test whether the ipsilateral
NSIs are able to affect the activity of contralateral
motoneurons. Current injections failed to elicit a
Journal of Neurobiology. DOI 10.1002/neu
Journal of Neurobiology. DOI 10.1002/neu
Depolarization
Depolarization
Depolarization
Depolarization
Depolarization
Hyperpolarization
Hyperpolarization
Hyperpolarization
Depolarization
Depolarization
E1 (3)
E2 (1)
E3 (4)
E4 (5)
E5/6 (5)
E7 (4)
E8 (5)
11 (6)
12 (2)
14 (4)
Depolarization
Depolarization
Depolarization
Depolarization
Hyperpolarization
Returns to
resting
potential
Returns to
resting
potential
Hyperpolarization
Depolarization
Depolarization
Ipsilateral
fCO Relaxation
Depolarization
Depolarization
Depolarization
Depolarization
Hyperpolarization*
Depolarization
Returns to resting
potential
Depolarization
No response
Depolarization
Depolarization
Depolarization
Hyperpolarization
No response
No response
Depolarization
Depolarization
Depolarization
Depolarization
Contralateral
fCO Relaxation
Depolarization
Contralateral
fCO Elongation
None
n.m.
None
None
None
Depolarization
(4/5 animals)
Hyperpolarization
None
No response
Hyperpolarization
Response to
Tonic Contralateral
fCO-Elongation
19.97 6 4.99
16.91 6 5.09
22.63 6 9.02
24.66 6 9.11
22.81 6 3.32
15.73 6 1.92
9.00 6 0.84
11.93 6 3.65
7.55 6 2.21
8.18 6 0.63
No response
8.49 6 0.98
n.m.
20.34 6 3.27
18.01 6 4.09
23.29 6 4.04
6.57 6 1.74
n.m.
7.54 6 2.21
12.47 6 2.62
Latency [ms]
to Contralateral
Stimulation
Latency [ms]
to Ipsilateral
Stimulation
fCO elongation/relaxation: velocity-sensitive response only. Tonic fCO elongation: position-sensitive response only. The average response latencies to ipsilateral fCO elongation are shown for E1,
E2, E4, E5/6, E7, E8, I1, I2, and I4. For E1, E4, E5/6, E7, E8, I1, I2, and I4 also the average response latencies to contralateral fCO elongation are given. n.m., not measured.
* The hyperpolarization of NSI E8 during contralateral fCO elongation was preceded by a short-latency depolarization.
Ipsilateral
fCO Elongation
NSI (# of
Recordings)
Table 2 Responses of Identified NSIs during Ramp-and-Hold fCO Elongation and Relaxation of the Ipsilateral and Contralateral fCOs and Number of Recordings
1262
Stein et al.
Interleg Couplings in the Stick Insect
1263
change in the activity of the contralateral extensor
motoneurons, regardless of the presence or absence
of PTX (N ¼ 14, not shown). Thus, although the NSIs
are part of the local circuit from sensors to motor activity, they do not appear to contribute to the transmission of sensory signals to the contralateral extensor motoneurons.
In the Presence of PTX, Stimulation
of the Contralateral fCO Influences
the Activity of Pro- and Retractor
Motoneurons
Previous investigations have shown that proprioceptive signals within a given segment do not only play a
role in intra-, but also in interjoint control (e.g., Hess
and Büschges, 1997, 1999; Akay et al., 2001). Therefore, we tested whether, in the presence of PTX, signals from the contralateral fCO also affected the
motoneurons of the other leg joints, for example, the
motoneurons supplying the thoraco-coxal joint. We
used a similar preparation as for the investigation of
the extensor tibiae motoneurons. We focused on the
extracellularly recorded response of the mesothoracic
motoneuron pools of the thoraco-coxal joint (pro- and
retractor motoneurons) while we stimulated the fCO
of the contralateral middle leg, with and without
PTX. In saline, stimulation of the contralateral fCO
did not elicit spike activity in the re- or protractor
motoneurons [Fig. 6(A)]. In general, no spike activity
was observed in either motoneuron pool in untreated
and inactive animals. Following application of PTX,
the pro- and retractor motoneurons exhibited long
phases of coactivation that could be interrupted by
episodes of strong bursting activity in both recordings, and in all tested animals (N ¼ 6 for retractor
nerve recordings, N ¼ 8 for protractor nerve recordings). When we stimulated the fCO of the contralateral leg during episodes of coactivation, the retractor
motoneurons responded with an increase in spike activity to elongation as well as relaxation of the fCO
[Fig. 6(B,C)]. While the combined activity of retractor motoneurons was always and in all experiments
related to the stimulus, the protractor motoneurons
showed stimulus-related activity in only two out of
eight tested animals [example shown in Fig. 6(B,D)].
To test whether the observed effects on the re- and
protractor motoneurons were due to nonspecific
mechanosensory influences rather than to a specific
influence from the contralateral fCO, we recorded the
activity of both motoneuron pools while we applied
tactile stimuli to the tarsus of the contralateral middle
leg in PTX. In none of our experiments (N ¼ 5) were
Figure 6 In PTX, sensory signals from the fCO affect
pro- and retractor motoneurons in the contralateral leg. (A)
Extracellular recordings of the retractor (nl5) and protractor
(nl2) motoneurons during contralateral fCO stimulation in
saline. (B) Extracellular recordings of retractor and protractor activity in PTX during an episode in which both motoneuron pools responded to contralateral fCO stimulation.
(C) PSTH of retractor and (D) of protractor motoneuronal
activity in PTX [same animal as in (A), n ¼ 4 ramps each,
BIN width 0.2 s].
the activities of the re- and protractor motoneurons
altered by these stimuli.
DISCUSSION
We characterized the influence of signals from the
sense organ that measures movements and positions
of the FT joint, the fCO, on the tibial motoneurons of
other legs in restrained animals. To establish the
transmission of fCO information we either activated
the animals or we bath applied PTX. When the locomotor system was active, fCO stimulation increased
the probability of clear-cut transitions in motor activJournal of Neurobiology. DOI 10.1002/neu
1264
Stein et al.
ity, from extensor to flexor activity and vice versa. In
PTX, signals from the middle leg fCO specifically
affected the activity of the motoneurons of the femurtibia joints of the other legs. The extensor and flexor
tibiae motoneurons of all legs received stimulus-dependent excitation. The responses to the ramp part of
fCO stimuli increased in a velocity-dependent manner. Tonic elongation of the fCO (which signals
flexed leg positions) caused an increase of extensor
activity in the ipsilateral fore and hind legs and
decreased extensor activity in all contralateral legs.
Tonically relaxed fCO positions caused opposite
effects. Our results show that position and movement
signals from the segmental fCO were transmitted
individually to the joint controllers of the adjacent
legs. By studying identified premotor nonspiking
interneurons of the contralateral leg, we show that
fCO signals were fed into the local networks known
to contribute to posture and movement control. The
NSIs showed different, but for each type specific,
responses. We conclude that the premotor nonspiking
interneurons process both local proprioceptive sensory signals and proprioceptive signals from other
legs.
Could Other Sense Organs Have
Contributed to the Observed
Intersegmental Influences via Indirect
Pathways?
The PTX experiments were performed with other
sense organs of the legs, for example hair plates, hair
rows, campaniform sensillae, and muscle receptors,
intact. It would thus be possible that, besides from
fCO signals, the activities of other sense organs could
have contributed to the changes observed in the activity of motoneurons in other legs. This is unlikely,
however, for the following reasons:
1. All interleg influences depended on fCO stimulus parameters (stimulus velocity or stimulus
position).
2. Sense organs measuring movements in the
other leg joints (summary: Bässler, 1983) could
not have been phasically activated because all
legs were tightly immobilized. Also, the receptor apodemes of all fCOs had been cut to prevent feedback from local fCOs.
3. Other sense organs in the femur of the stimulated leg could not have been activated either,
in particular, multipolar sensory cells, apodeme-receptors/tension-receptors and musclereceptor-organs (Bässler, 1977), and strandreceptors (Bräunig, 1982; Pflüger and Burrows,
Journal of Neurobiology. DOI 10.1002/neu
1987), as well as leg campaniform sensilla
(Schmitz, 1993). This is because extensor and
flexor muscles and nerves had been cut and no
forces could act on these sense organs.
4. Finally, a contribution of indirect sensory pathways to the observed influences in PTX via
stimulus-related activation of leg muscles is
unlikely because the latencies of the responses
of the contralateral NSIs and extensor tibiae
motoneurons were in the range of 10–20 ms
(Table 2) and thus too short to include indirect
pathways affording muscle activation. The
latencies of the motoneuronal responses
observed in experiments with active animals
were considerably longer.
Thus, in these experiments, a contribution of indirect
effects via the activation of muscles cannot be
excluded. There are, however, several reasons why
the latencies in active animals might differ from those
of the PTX experiments. In the PTX experiments the
onset of the de- or hyperpolarizing synaptic inputs in
the neuropil region of inter- and motoneurons was
used to measure the latency, while in active animals
the first spike response on the spatially distant extracellular recording was used instead. Besides the time
that the motoneuronal action potential needs to propagate to the extracellular recording site, synaptic
inputs to the motoneurons have to summate prior to
burst onset. Furthermore, in active animals, sensory
influences from the contralateral leg have to interact
with the activity of rhythmically active local, segmental neural networks (cf. Büschges, 2005) in the
contralateral leg so that a longer latency of the motor
response seems conceivable.
Comparison with Other Systems
In general, intersegmental coordination of movements in locomotor systems producing a rhythmic
output can result from an exchange of information
between central elements of the nervous system, such
as segmental central pattern generators (e.g., Grillner
et al., 1991; Matsushima and Grillner, 1992; Kristan
and Calabrese, 1976; Friesen, 1989; Friesen and
Pearce, 1993; Ikeda and Wiersma, 1964; Sillar et al.,
1987; Ryckebusch and Laurent, 1994), by an
exchange of segmental sensory information (e.g.,
Ritzmann et al., 1991; Sillar et al., 1987; Nagayama
et al., 1993; Lansner and Ekeberg, 1994; Laurent,
1991), or by a combination of both (reviews in:
Büschges, 2005; Grillner and Wallén, 2002; Hill
et al., 2003). An example for the latter is the influence
Interleg Couplings in the Stick Insect
of the thoracic-coxal muscle receptor organ on the
motoneurons of other ipsilateral legs in crustaceans.
A suprathreshold activation of the motoneurons is
only achieved in rhythmic preparations when a central pattern generator is active (Sillar et al., 1987).
Similarly, the processing of sensory signals in the
middle leg and the ipsilateral hind leg of the locust
appears to be subject to central influences (summary:
Laurent, 1991). In general, the neuronal layout of the
locust is considered to be very similar to stick insects
(e.g., Burrows, 1996; Büschges et al., 2000; Field and
Matheson, 1998). Intracellular recordings from intersegmental interneurons in the locust show that some
of these neurons respond to active movements of the
tibia and to sensory signals from different sense
organs in the middle leg (Laurent, 1986, 1987). They
synapse onto local premotor interneurons and motoneurons of the hind leg (Laurent and Burrows, 1989).
This shows that the local premotor network is, in
principle, capable of processing sensory signals from
other legs. Nonspiking interneurons seem to play a
crucial role in this processing, because Laurent and
Burrows (1989) did not observe any synapses
between intersegmental interneurons and local spiking interneurons. In these experiments, the sensory
information carried by the intersegmental interneurons was not sufficient to elicit suprathreshold activation of motoneurons. Laurent (1986) hypothesized
that, for a spike response of the motoneurons, an
additional general arousal needs to be present and
supply drive to the motoneurons. PTX or an activation of the animal by disturbances might have exactly
that effect. Because the intersegmental interneurons
receive GABA-ergic inhibition from local spiking
interneurons in the mesothoracic ganglion (Laurent,
1988; Watson and Laurent, 1990), blocking these
inputs with PTX could reduce this inhibition and thus
establish a more effective transmission of sensory information to the hind legs, as is needed during coordinated leg movements.
Local Processing of Contralateral and
Intersegmental Sensory Signals
How sensory information from the fCO of a different
leg is processed by the local leg control system was
studied in the mesothoracic segment. Identified local
nonspiking interneurons, which are known to process
sensory signals from the local fCO, contribute to excitation of the extensor motoneurons in response to contralateral fCO stimulation. In PTX, most NSIs received
depolarizing input, for example, E1, E3, E4, E5/6, and
E7. These synaptic inputs appeared only after PTX
1265
application, thus they could not have been mediated
by PTX-sensitive chloride channels. Interneurons E4
and I4 showed a response that was stronger than during stimulation of the ipsilateral fCO. All responding
NSIs received velocity-dependent inputs, while only a
subset of NSIs was affected by stimulus position, for
example, E1, E5/6, and E7. Thus, similar to the processing of local fCO signals, individual NSIs showed
unique responses, which depended on both stimulus
velocity and position. Some of the identified premotor
NSIs showed synaptic inputs that would support the
activity of the tibial motoneurons, as judged from their
synaptic contacts to the motoneurons, while others
would antagonize it. Thus, the processing of sensory
signals from other legs shares further similarities with
the processing of segmental sensory signals (femurtibia joint: Bässler, 1993; thoraco-coxal joint:
Büschges and Schmitz, 1991), in the form of distributed processing of proprioceptive signals (see also
Bässler, 1993; Büschges et al., 2000).
Functional Significance of the Observed
Information Flow between the Joint
Controllers of Different Legs
During walking, coordinating influences between legs
adjust the action of the adjacent legs towards a functional gait for locomotion (summary: Cruse, 1990). In
those experiments, however, the specific influence of a
sense organ in one leg on the motoneuronal activity in
the other legs was not investigated in detail, with few
exceptions (Graham and Wendler, 1981; Ludwar
et al., 2005). This is mainly due to: (i) the complex activity of motoneurons and sensory neurons of different
leg sense organs in the active animal, and (ii) the
subtle influence that signals from a single, individual
sense organ can exert in the concerted action of a
walking animal (cf. Bässler and Büschges, 1998).
When the locomotor system was activated in our
experiments, ramp-and-hold stimulation of the fCO
facilitated transitions in the motor output of the contralateral extensor-flexor system. Interestingly, vibration stimuli to the ipsilateral fCO (Sauer and Stein,
1999; Bässler et al., 2003) elicit similar responses in
the ipsilateral tibial motoneurons. In both experimental situations fCO stimuli are capable of synchronizing state transitions, independent of their direction,
and could thus be used to facilitate stance-swing or
swing-stance transitions in neighboring legs during
active leg movements.
In restrained, inactive animals the impact of leg
proprioceptors on the muscles of other legs is either
absent or diminished. The motor outputs of the differJournal of Neurobiology. DOI 10.1002/neu
1266
Stein et al.
ent joint control systems and the activities of the proprioceptive leg sense organs are in a steady state
(e.g., Bässler, 1983). We used these advantages to
study how sensory information from other leg proprioceptors is processed in the local FT joint control
system. GABA-ergic inhibition seems to be ubiquitously present in the thoracic nerve cord of insects
(e.g., locust, Watson, 1986; Wiens and Wolf, 1993;
Watson and Laurent, 1990) and thus appeared as a
good candidate for underlying a possible diminishment of pathways between the joint control systems
of different legs. We tested this hypothesis with bath
application of PTX. PTX reduces the amount of inhibition in the FT joint control network (Sauer et al.,
1997) as is assumed to happen when an animal
switches from an inactive to an active state. When the
descending pathways from the supraesophageal ganglion to the thoracic ganglia are severed, the activity
of tibial motoneurons increases and the animal starts
to perform active leg movements (Eidmann, 1956;
Graham, 1979). Changes in the amount of inhibition
also seem to occur during active states in locusts
(Wolf and Burrows, 1995). PTX does not affect the
membrane properties of local NSIs and motoneurons
(Sauer et al., 1997), but it enhances the transmission
of sensory information from the fCO to these neurons. We show that PTX also enhances interleg couplings. In PTX, sensory signals from the middle leg
fCO strongly affected the activity of tibial motoneurons innervating the legs of all other segments. This
showed the action of intra- and intersegmental pathways capable of transmitting specific sensory information from one joint sensor to the control systems
of the same leg joint of other legs. Velocity and position signals were processed by the interaction of
antagonistic and parallel pathways at the level of the
premotor nonspiking interneurons. It is quite conceivable that in actively moving animals, for example,
during walking or rocking (Bässler and Wegner,
1983; Ludwar et al., 2005; Pflüger, 1977), the same
layer of interneurons processes sensory information
from the fCOs of other legs. After all, the local
NSIs play an important role in the generation of such
walking movements (Bässler and Büschges, 1998;
Driesang and Büschges, 1996).
In the presence of PTX, sinusoidal stimulation of
the middle leg fCO elicited tibial forces that would
lead to an approximate antiphase movement of the ipsilateral and contralateral legs in the pro- and metathoracic segments (Fig. 2). Several recordings additionally indicated a phase shift between the force
maxima of the left legs and between the movements
of the right legs, respectively. This finding was not
investigated further. In general, the muscles of the
Journal of Neurobiology. DOI 10.1002/neu
contralateral middle leg were activated in phase with
the imposed movement (but in antiphase with the
motoneuronal activity of this leg). This situation differs from the coordination observed during walking,
which, in the adult stick insect, is usually a tetrapod
gait (Graham and Cruse, 1981; Epstein and Graham,
1983). In this gait antiphase activity is confined to the
brief periods of return strokes in adjacent legs. It is,
however, not surprising that the responses observed
in the motoneurons with PTX differ from those in
walking animals, because the local NSIs contribute
to the generation of walking movements (Bässler
and Büschges, 1998; Driesang and Büschges, 1996).
Thus, if the NSIs indeed are the main gates for signal
transmissions from the contralateral fCO (or from the
fCOs of other legs in general), the processing of local
fCO signals during active walking (and thus actively
moving tibia) would differ from that in our experiments. Here, all FT control loops remained in an
inactive state and sensory feedback from all legs was
eliminated, that is, the NSIs were specifically affected
by the activity of the contralateral fCO, without interference from other sense organs or centrally generated patterns.
Rather than being related to walking, the motoneuronal activity of all legs corresponded to rocking
behavior (Pflüger, 1977). Rocking supports a basic
behavior of the stick insect, the twig mimesis
(Bässler, 1983). During rocking, the tibiae of all ipsilateral legs move in antiphase with the tibiae of all
contralateral legs. The legs are not lifted off the
ground. This creates a swinging movement of the
body. Similar to the experimental situation in PTX,
the FT control systems remain in an inactive state,
generating resistance reflexes during this behavior,
and antiphase activity is present throughout the whole
cycle of leg movement.
In our experiments, we used PTX to remove
GABA-ergic inhibition en masse in the thoracic nerve
chord, a situation unlikely to occur when the animal
starts to move. The extensive removal of inhibition
served us as a tool to reveal active, possibly excitatory, connections between the joint controllers of the
different legs. In untreated, but actively moving animals, it seems rather likely that, instead of a massive
suppression of inhibition, specific inhibitory pathways are diminished while excitatory pathways may
be enhanced. Such effects could be due to local influences or pathways descending from higher neuronal
centers and would lead to a similar result as an application of PTX in resting animals, namely a shift in
the balance between excitation and inhibition towards
a stronger influence of excitatory pathways. Future
experiments focusing on the effects of excitatory con-
Interleg Couplings in the Stick Insect
nections between the joint controllers of the different
legs may thus lead towards further understanding of
leg coordination in actively moving animals.
We would like to thank Arne Sauer and Harald Wolf for
their support and their comments on the article.
REFERENCES
Akay T, Bässler U, Gerharz P, Büschges A. 2001. Interjoint
coordination: The role of sensory signals from the coxatrochanteral joint on controlling motoneuronal activity of
the femur-tibia joint in the stick insect. J Neurophysiol
85:594–604.
Bässler U. 1977. Sense organs in the femur of the stick
insect and their relevance to the control of position of the
femur-tibia-joint. J Comp Physiol A 121:99–113.
Bässler U. 1983. Neural basis of elementary behavior in
stick insects. Berlin: Springer Verlag. 169 p.
Bässler U. 1993. The femur-tibia control system of stick
insects—a model system for the study of the neural basis
of joint control. Brain Res Rev 18:207–226.
Bässler U, Büschges A. 1998. Pattern generation for stick
insect walking movements—multisensory control of a
locomotor program. Brain Res Rev 27:65–88.
Bässler U, Sauer AE, Büschges A. 2003. Vibration signals
from the FT joint can induce phase transitions in both
directions in motoneuron pools of the stick insect walking system. J Neurobiol 56:125–138.
Bässler U, Wegner U. 1983. Motor output of the denervated
thoracic ventral nerve cord in the stick insect Carausius
morosus. J Exp Biol 105:127–145.
Bräunig P. 1982. Strand receptors with central cell bodies
in the proximal leg joints of orthopterous insects. Cell
Tissue Res 222:647–654.
Brunn DE, Dean J. 1994. Intersegmental and local interneurons in the metathorax of the stick insect Carausius
morosus that monitor leg position. J Neurophysiol 72:
1208–1219.
Burrows M. 1996. The Neurobiology of an insect brain.
Oxford: Oxford University Press. 682 p.
Büschges A. 1989. Processing of sensory input from the
femoral chordotonal organ by spiking interneurones of
the stick insect. J Exp Biol 144:81–111.
Büschges A. 1990. Non-spiking pathways in a joint-control
loop of the stick insect Carausius morosus. J Exp Biol
151:133–160.
Büschges A. 1994. The physiology of sensory cells in the
ventral scoloparium of the stick insect femoral chordotonal organ. J Exp Biol 189:285–292.
Büschges A. 2005. Sensory control and organization of
neural networks mediating coordination of multisegmental organs for locomotion. J Neurophysiol 93:1127–1135.
Büschges A, Kittmann R, Schmitz J. 1994. Identified nonspiking interneurons in leg reflexes and during walking
in the stick insect. J Comp Physiol A 174:685–700.
Büschges A, Sauer AE, Bässler U. 2000. Flexibility of a
1267
Proprioceptive Feedback System Results from its ‘‘Parliamentary’’ (Distributed) Organization. In: Cruse H,
Dean J, Ritter H, editors. Prerational Intelligence: Adaptive Behavior and Intelligent Systems Without Symbols
and Logic. Volume I. Netherlands: Kluwer Academic
Publishers, p 267–286.
Büschges A, Schmitz J. 1991. Nonspiking pathways opposing the resistance reflex in the subcoxal joint of stick
insects. J Neurobiol 22:224–237.
Cruse H. 1990. What mechanisms coordinate leg movement
in walking arthropods. TINS 13:15–21.
Cruse H, Brunn D, Bartling C, Dean J, Dreifert M, Kindermann T, Schmitz J. 1995. Walking—a complex behavior
controlled by simple networks. Adaptive Behavior 3:385–
418.
Cruse H, Schmitz J, Braun U, Schweins A. 1993. Control of
body height in the stick insect walking on a treadwheel.
J Exp Biol 181:141–155.
Dixon WJ, Massey FJ. 1969. Introduction to statistical analysis.3rd edition. New York: McGraw Hill.488 p.
Dörr H, Heß D, Gramoll S. 1996. Interstitial voltage and
potassium concentration in the mesothoracic ganglion of
a stick insect at rest and during neuronal activation.
J Insect Physiol 42:967–974.
Driesang RB, Büschges A. 1993. The neural basis of catalepsy in the stick insect. IV. Properties of nonspiking
interneurons. J Comp Physiol A 173:445–454.
Driesang RB, Büschges A. 1996. Physiological changes in
central neuronal pathways contributing to the generation
of reflex reversal. J Comp Physiol A 179:45–57.
Dürr V, Schmitz J, Cruse H. 2004. Behaviour-based
modelling of hexapod locomotion: Linking biology
and technical application. Arthropod Struct Dev 33:237–
250.
Eidmann H. 1956. Über rhythmische Erscheinungen bei der
Stabheuschrecke Carausius morosus (Br.). Z Vgl Physiol
38:370–390.
Epstein S, Graham D. 1983. Behavior and motor output for
an insect walking on a slippery surface. I: forward walking. J Exp Biol 105:215–229.
Field L, Matheson T. 1998. Chordotonal organs in insects.
Adv Insect Physiol 27:1–228.
Friesen WO. 1989. Neuronal control of leech swimming
movements. In: Jacklet JW, editor. Cellular and Neuronal Oscillators. New York: Dekker, p.269–316.
Friesen WO, Pearce RA. 1993. Mechanisms of intersegmental coordination in leech locomotion. Semin Neurosci 5:41–47.
Graham D. 1979. Effects of circum-oesophageal lesion on
the behavior of the stick insect Carausius morosus. I.
Cyclic behavior patterns. Biol Cybern 32:139–145.
Graham D, Cruse H. 1981. Coordinated walking of stick
insects on a mercury surface. J Exp Biol 92:229–241.
Graham D, Wendler G. 1981. Motor output to the protractor
and retractor coxae muscles in stick insects walking on a
treadwheel. Physiol Entomol 6:161–174.
Grillner S, Wallén P. 2002. Cellular bases of a vertebrate
locomotor system-steering, intersegmental and segmental co-ordination and sensory control. Brain Res Rev 40:
Journal of Neurobiology. DOI 10.1002/neu
1268
Stein et al.
92–106.
Grillner S, Wallén P, Brodin L, Lanser A. 1991. Neuronal
network generating locomotor behavior in lamprey: circuitry, transmitters, membrane properties, and simulation. Annu Rev Neurosci 14:169–199.
Hess D, Büschges A. 1997. Sensorimotor pathways involved in interjoint reflex action of an insect leg. J Neurobiol
33:891–913.
Hess D, Büschges A. 1999. Role of proprioceptive signals
from the stick insect femur-tibia joint in patterning motoneuronal activity of an adjacent leg joint. J Neurophysiol
81:1856–1865.
Hill AAV, Masino MA, Calabrese RL. 2003. Intersegmental Coordination of Rhythmic Motor Patterns. J Neurophysiol 90:531–538.
Hofmann T, Koch UT, Bässler U. 1985. Physiology of the
femoral chordotonal organ in the stick insect, Cuniculina
impigra. J Exp Biol 114:207–223.
Ikeda K, Wiersma CA. 1964. Autogenic rhythmicity in the
abdominal ganglia of the crayfish: the control of swimmeret movements. Comp Biochem Physiol 12:107–115.
Kittmann R, Schmitz J, Büschges A. 1996. Premotor interneurons in generation of adaptive leg reflexes and voluntary movements in stick insects. J Neurobiol 31:512–532.
Kristan WB Jr., Calabrese RL. 1976. Rhythmic swimming
activity in neurones of the isolated nerve cord of the
leech. J Exp Biol 65:643–688.
Lansner A, Ekeberg Ö. 1994. Neuronal network models of
motor generation and control. Curr Opin Neurobiol 4:903–
908.
Laurent G. 1986. Thoracic intersegmental interneurones in
the locust with mechanoreceptive inputs from a leg.
J Comp Physiol A 159:171–186.
Laurent G. 1987. The morphology of a population of thoracic intersegmental interneurones in the locust. J Comp
Neurol 256:412–429.
Laurent G. 1988. Local circuits underlying excitation and
inhibition of intersegmental interneurones in the locust.
J Comp Physiol A 162:145–157.
Laurent G. 1991. Intersegmental interneurones and interactions between local centres of integration in the locust
central nervous system. In: Armstrong BMH, Bush DM,
editors. Locomotor neural mechanisms in arthropods and
vertebrates. Manchester, UK: Manchester University Press,
p 11–24.
Laurent G, Burrows M. 1989. Intersegmental interneurons
can control the gain of reflexes in adjacent segments of
the locust by their action on nonspiking local interneurons. J Neurosci 9:3030–3039.
Ludwar BC, Göritz M, Schmidt J. 2005. Intersegmental
Coordination of Walking Movements in Stick Insects.
J Neurophysiol 93:1255–1265.
Matsushima T, Grillner S. 1992. Neural mechanisms of
intersegmental coordination in lamprey: local excitability
changes modify the phase coupling along the spinal cord.
J Neurophysiol 67:373–388.
Nagayama T, Isogai Y, Sato M, Hisada M. 1993. Intersegmental ascending interneurons controlling uropod movements of the crayfish Procambarus clarkii. J Comp NeuJournal of Neurobiology. DOI 10.1002/neu
rol 332:155–174.
Newland PL. 1990. Morphology of a population of mechanosensory ascending interneurones in the metathoracic
ganglion of the locust. J Comp Neurol 299:242–260.
Orlovsky GN, Deliagina TG, Grillner S. 1999. Neuronal
control of locomotion from mollusc to man. New York:
Oxford University Press. p 5–116.
Pearson KG. 1995. Proprioceptive regulation of locomotion. Curr Opin Neurobiol 5:786–791.
Pflüger H-J. 1977. The control of the rocking movements of
the phasmid Carausius morosus Br J Comp Physiol 120:
181–202.
Pflüger H-J, Burrows M. 1987. A strand receptor with a
central cell body synapses upon spiking local interneurones in the locust. J Comp Physiol A 160:295–304.
Ritzmann RE, Pollack AJ, Hudson SE, Hyvonen A. 1991.
Convergence of multi-modal sensory signals at thoracic
interneurons of the escape system of the cockroach, Periplaneta americana. Brain Res 563:175–183.
Ryckebusch S, Laurent G. 1994. Interactions between segmental leg central pattern generators during fictive
rhythms in the locust. J Neurophysiol 72:2771–2785.
Sauer AE, Büschges A, Stein W. 1997. The role of presynaptic inputs to proprioceptive afferents in tuning sensorimotor pathways of an insect joint-control network.
J Neurobiol 32:359–376.
Sauer AE, Driesang RB, Büschges A, Bässler U. 1995. Information processing in the femur-tibia control loop of
stick insects 1. The response characteristics of two nonspiking interneurons result from parallel excitatory and
inhibitory inputs. J Comp Physiol A 177:145–158.
Sauer AE, Driesang RB, Büschges A, Bässler U. 1996.
Distributed processing on the basis of parallel and antagonistic pathways. Simulation of the femur-tibia control system in the stick insect. J Comput Neurosci 3:179–
198.
Sauer AE, Stein W. 1999. Sensorimotor pathways processing vibratory signals from the femoral chordotonal organ
of the stick insect. J Comp Physiol A 185:21–31.
Schmitz J. 1993. Load compensatory reactions in the proximal leg joints of stick insects during standing and walking. J Exp Biol 183:15–33.
Schmitz J, Delcomyn F, Büschges A. 1991. Oil and hook
electrodes for en passant recording from small nerves. In:
Conn PM, editor. Methods in Neuroscience 4. San Diego:
Academic Press, p 266–278.
Sillar KT, Clarac F, Bush BMH. 1987. Intersegmental coordination of central neural oscillators for rhythmic movements of the walking legs in crayfish, pacifastacus
leniusculus. J Exp Biol 131:245–264.
Stein W, Sauer AE. 1998. Modulation of sensorimotor pathways associated with gain changes in a posture-control
network of an insect. J Comp Physiol A 183:489–501.
Treherne JE. 1985. Blood-brain barrier. In: Kerkut GA,
Gilbert LI, editors. Comprehensive Insect Physiology,
Biochemistry and Pharmacology, Vol. 5. Oxford: Pergamon Press, p 115–137.
Watson AHD. 1986. The distribution of GABA-like immunoreactivity in the thoracic nervous system of the locust
Interleg Couplings in the Stick Insect
Schistocerca gregaria. Cell Tissue Res 246:331–341.
Watson AHD, Laurent G. 1990. GABA-like immunoreactivity in a population of locust intersegmental interneurones and their inputs. J Comp Neurol 302:761–767.
Weidler DJ, Diecke FPJ. 1969. The role of cations in conduction in the central nervous system of the herbivorous
insect Carausius morosus. Z vergl Physiol 64:372–399.
Weiland G, Bässler U, Brunner M. 1986. A biological feedback control system with electronic input: The artificially
closed femur-tibia control system of stick insects. J Exp
Biol 120:369–385.
1269
Weiland G, Koch UT. 1987. Sensory feedback during
active movements of stick insects. J Exp Biol 133:137–
156.
Wiens TJ, Wolf H. 1993. The inhibitory Motoneurons of
Crayfish Thoracic Limbs: Identification, Structures and
Homology With Insect Common Inhibitors. J Comp Neurol 336:216–178.
Wolf H, Burrows M. 1995. Proprioceptive sensory neurons
of a locust leg receive rhythmic presynaptic inhibition
during walking. J Neurosci 15:5623–5636.
Journal of Neurobiology. DOI 10.1002/neu